Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that β-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4+ and CD8+ T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca2+) influx, and that either the intracellular Ca2+ chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca2+-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

Galectin-9 expression was examined in 6 human melanoma cell lines. Among them, MM-BP proliferated with colony formation, but MM-RU failed. RT-PCR analysis revealed evident expression of galectin-9 mRNA in MM-BP but not in MM-RU. MM-BP expressed galectin-9 protein both on the surface and in the cytoplasm, whereas MM-RU expressed it only weakly in the cytoplasm. Exogenous galectin-9 induced in vitro both cell aggregation and apoptosis of MM-RU proliferating without colony formation. Association of galectin-9 expression in melanoma cells with prognosis of the patients bearing melanocytic tumors was further examined. Galectin-9 protein was strongly and homogeneously expressed in melanocytic nevi, but down-regulated in melanoma cells especially in metastatic lesions. High galectin-9 expression was inversely correlated with the progression of this disease, suggesting that high galectin-9 expression in primary melanoma lesions links to a better prognosis.

Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-γ stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-γ up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-γ up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-γ up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-γ-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-γ-stimulated fibroblasts as a physiological modulator at the inflammatory sites.

Little is known about the roles of galectins, a family of beta-galactoside-binding lectins, in myeloid cell differentiation. In the present experiments, we used HL-60 cells as a model of myeloid cell differentiation. The HL-60 cells were differentiated into eosinophil-, monocyte-, and neutrophil-like cells by coculture with sodium butyrate under a mild alkaline condition, phorbol 12-myristate 13-acetate, and dimethyl sulfoxide, respectively. Thus, the expression of galectins in HL-60 cells during differentiation into three different lineages was assessed. Reverse transcriptase-polymerase chain reaction analyses revealed that undifferentiated HL-60 cells expressed galectin-1, -3, -8, -9, and -10 (identical to Charcot Leyden crystal) mRNAs, and galectin-2, -4, and -7 were negligible before and after the differentiations. We failed to detect evident changes in the mRNA levels of galectin-1 and -8 during the differentiations. However, during the eosinophilic differentiation, galectin-9 mRNA expression was gradually decreased, whereas galectin-10 mRNA expression was increased. During the course of monocytic differentiation, galectin-9 mRNA expression was down-regulated, whereas galectin-3 mRNA expression was up-regulated. Moreover, only galectin-10 mRNA expression was enhanced in the process of neutrophilic differentiation. These changes in galectin expressions were confirmed by Western blot and flow cytometry analyses. It is thus suggested that changes in the expressions of galectin-3, -9, and -10 are potentially important for myeloid cell differentiation into specific lineages.